U.S. patent number 10,227,332 [Application Number 15/518,473] was granted by the patent office on 2019-03-12 for t-type calcium channel modulator and uses thereof.
This patent grant is currently assigned to THE UNIVERSITY OF MONTANA, UTI LIMITED PARTNERSHIP. The grantee listed for this patent is University of Montana, UTI Limited Partnership. Invention is credited to Philippe Diaz, Gerald Werner Zamponi.
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United States Patent |
10,227,332 |
Zamponi , et al. |
March 12, 2019 |
T-type calcium channel modulator and uses thereof
Abstract
The present invention provides T-type calcium channel modulators
and methods for producing and using the same. In some embodiments,
compounds of the invention are of the formula: ##STR00001## where
R.sup.1 is selected from the group consisting of alkyl, alkenyl,
polyether, alkoxy and cycloalkyl; X is selected from the group
consisting of methylene, --C(.dbd.O)--[NR.sup.4].sub.a--,
--C(.dbd.S)--, and --S(.dbd.O).sub.b; R.sup.2 is selected from the
group consisting of heterocycloalkyl and heteroaryl; R.sup.3 is
selected from the group consisting of alkyl, alkenyl, polyether,
alkoxy, cycloalkyl, --NR.sup.5R.sup.6, --C(.dbd.O)NR.sup.5R.sup.6,
--C(.dbd.O)OR.sup.a (where R.sup.a is alkyl, typically
C.sub.1-C.sub.8 alkyl, often C.sub.2-C.sub.6 alkyl, and in one
particular embodiment R.sup.a is tert-butyl), and
--SO.sub.2NR.sup.5R.sup.6; R.sup.4 is hydrogen, alkyl, or a
nitrogen protecting group; each of R.sup.5 and R.sup.6 are
independently selected from the group consisting of hydrogen and
alkyl; a is 0 or 1; b is 1 or 2; n=1 to 3; and m=0 to 1.
Inventors: |
Zamponi; Gerald Werner
(Calgary, CA), Diaz; Philippe (Missoula, MT) |
Applicant: |
Name |
City |
State |
Country |
Type |
UTI Limited Partnership
University of Montana |
Calgary
Missoula |
N/A
MT |
CA
US |
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|
Assignee: |
UTI LIMITED PARTNERSHIP
(Calgary, Alberta, CA)
THE UNIVERSITY OF MONTANA (Missoula, MT)
|
Family
ID: |
55909609 |
Appl.
No.: |
15/518,473 |
Filed: |
October 15, 2015 |
PCT
Filed: |
October 15, 2015 |
PCT No.: |
PCT/US2015/055757 |
371(c)(1),(2),(4) Date: |
April 11, 2017 |
PCT
Pub. No.: |
WO2016/073160 |
PCT
Pub. Date: |
May 12, 2016 |
Prior Publication Data
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|
|
Document
Identifier |
Publication Date |
|
US 20170233374 A1 |
Aug 17, 2017 |
|
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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62064364 |
Oct 15, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07D
403/12 (20130101); C07D 209/88 (20130101); C07D
401/12 (20130101); Y02P 20/55 (20151101) |
Current International
Class: |
C07D
209/88 (20060101); C07D 403/12 (20060101); C07D
401/12 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Chemical Abstracts Registry No. 1007567-83-0, indexed in the
Registry file on STN CAS Online on Mar. 12, 2008. cited by examiner
.
Chemical Abstracts Registry No. 1492851-81-6, indexed in the
Registry file on STN CAS Online Dec. 11, 2013. cited by examiner
.
Chemical Abstracts Registry No. 53905-55-8, indexed in the Registry
file on STN CAS Online Nov. 16, 1984. cited by examiner .
Chemical Abstracts Registry No. 1380048-66-7, indexed in the
Registry file on STN CAS Online Jun. 25, 2012. cited by
examiner.
|
Primary Examiner: Stockton; Laura L
Attorney, Agent or Firm: Cha; Don D. Hamilton DeSanctis
& Cha, LLP
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the priority benefit of U.S. Provisional
Application No. 62/064,364, filed Oct. 15, 2014, which is
incorporated herein by reference in its entirety.
Claims
What is claimed is:
1. A compound of the formula: ##STR00026## wherein R.sup.1 is
alkyl; X is selected from the group consisting of
--C(.dbd.O)--[NR.sup.4].sub.a--, --C(.dbd.S)--, and
--S(.dbd.O).sub.b--; R.sup.2 is selected from the group consisting
of piperidinyl and pyrrolidinyl; R.sup.3 is selected from the group
consisting of --C(.dbd.O)NH-tBu and 3,3-dimethylbutyl; R.sup.4 is
hydrogen, alkyl, or a nitrogen protecting group; a is 0 or 1; b is
1 or 2; n=1 to 3; and m=0 to 1.
2. The compound of claim 1, wherein R.sup.1 is selected from the
group consisting of C.sub.1-C.sub.10 alkyl.
3. The compound of claim 2, wherein R.sup.1 is propyl, butyl, or
pentyl.
4. The compound of claim 1, wherein X is --C(.dbd.O)--NH--.
5. The compound of claim 4, wherein n is 1.
6. The compound of claim 1, wherein R.sup.2 is selected from the
group consisting of ##STR00027##
7. The compound of claim 4, wherein m is 1.
8. The compound of claim 4, wherein R.sup.3 is
--C(.dbd.O)NH-tBu.
9. The compound of claim 8, wherein R.sup.3 is
3,3-dimethylbutyl.
10. The compound of claim 1, wherein R.sup.1 is selected from the
group consisting of propyl, butyl and pentyl.
11. A method for treating a clinical condition associated with
T-type calcium channel activation, said method comprising
administering to a subject in need of such a treatment a
therapeutically effective amount of a compound of claim 1 wherein
treating does not embrace preventing.
12. The method of claim 11, wherein said clinical condition
associated with T-type calcium channel activation comprises acute
inflammatory pain, tactile allodynia, diabetic neuropathy,
pulmonary hypertension, chemotherapeutic induced neuropathy,
chronic pain, diabetes, epilepsy, visceral pain, cancer pain,
cardiac hypertrophy, or a combination thereof.
13. A method for treating a clinical condition associated with
T-type calcium channel activation, said method comprising
administering to a subject in need of such a treatment a
therapeutically effective amount of a compound selected from the
group consisting of: ##STR00028## ##STR00029## wherein treating
does not embrace preventing.
Description
FIELD OF THE INVENTION
The present invention relates to T-type calcium channel modulators
and methods for producing and using the same. In some embodiments,
compounds of the invention also have analgesic effects.
BACKGROUND OF THE INVENTION
T-type calcium channels are known for regulating neuronal and
cardiac pacemaker activity. They open in response to small membrane
depolarizations that in turn trigger the initiation of action
potentials[1]. Disruption of this sensitive signaling mechanism
often leads to hyperexcitability disorders such as arrhythmia,
epilepsy and pain[2-8]. Similarly, up-regulation of T-type channels
in primary afferent fibers has been linked to chronic pain
disorders, whereas ablation of these channels mediates analgesia[3,
4, 7]. The development of new selective T-type channel antagonists
has not been a trivial undertaking, with only a few such small
organic molecules having recently been identified[5].
Therefore, there is a continuing need for compounds that can
modulate T-type calcium channels.
SUMMARY OF THE INVENTION
Low-voltage-activated (T-type) calcium channels are important
regulators of the transmission of nociceptive information in the
primary afferent pathway and finding ligands (e.g., compounds) that
modulate these channels is a key focus of the drug discovery field.
Some aspects of the invention provide T-type calcium channel
modulators. In some embodiments, compounds of the invention exhibit
mixed cannabinoid receptor/T-type channel blocking activity. In
other embodiments, compounds of the invention also exhibit
analgesic effects in treatment of pain. Still in other embodiments,
compounds of the invention exhibit selective Cav3.2 inhibitors
without substantially influencing cannabinoid receptor activity.
That is, compounds of the invention have EC.sub.50 cannabinoid
receptor activity of about 50 .mu.M or more, typically about 100
.mu.M or more, often about 250 .mu.M or more and more often about
500 .mu.M or more.
One particular aspect of the invention provides a compound of the
formula:
##STR00002## where R.sup.1 is selected from the group consisting of
alkyl, alkenyl, polyether, alkoxy and cycloalkyl; X is selected
from the group consisting of methylene,
--C(.dbd.O)--[NR.sup.4].sub.a, --C(.dbd.S)--, and
--S(.dbd.O).sub.b--; R.sup.2 is selected from the group consisting
of heterocycloalkylene and heteroarylene; R.sup.3 is selected from
the group consisting of alkyl, alkenyl, polyether, alkoxy,
cycloalkyl, --NR.sup.5R.sup.6, --C(.dbd.O)NR.sup.5R.sup.6,
--C(.dbd.O)--OR.sup.a (where R.sup.a is alkyl, typically
C.sub.1-C.sub.8 alkyl, often C.sub.2-C.sub.6 alkyl, and in one
particular embodiment R.sup.a is tert-butyl), and
--SO.sub.2NR.sup.5R.sup.6; R.sup.4 is hydrogen, alkyl, or a
nitrogen protecting group; each of R.sup.5 and R.sup.6 are
independently selected from the group consisting of hydrogen and
alkyl; a is 0 or 1; b is 1 or 2; n=1 to 3; and m=0 to 1.
As used herein, "alkyl" refers to a saturated linear monovalent
hydrocarbon moiety of one to twenty, typically one to ten, and
often two to eight carbon atoms or a saturated branched monovalent
hydrocarbon moiety of three to twenty, typically three to ten, and
often three to eight carbon atoms. Exemplary alkyl group include,
but are not limited to, methyl, ethyl, n-propyl, 2-propyl,
tert-butyl, pentyl, and the like. The term "alkenyl" refers to a
linear monovalent hydrocarbon moiety of two to twenty, typically
two to ten, and often two to eight carbon atoms or a branched
monovalent hydrocarbon moiety of three to twenty, typically three
to ten, and often three to eight carbon atoms having at least one
carbon-carbon double bond. The term "alkoxy" means a moiety --OR
where R is an alkyl as defined herein. The terms "heterocyclyl" and
"heterocycloalkyl" are used interchangeably herein and refer to a
non-aromatic monocyclic moiety of three to eight ring atoms in
which one or more, typically one or three, and often one or two
ring atoms are heteroatoms selected from N, O, or S(O).sub.n (where
n is an integer from 0 to 2), the remaining ring atoms being C,
where one or two C atoms can optionally be a carbonyl group. The
heterocyclyl ring can be optionally substituted independently with
one or more, preferably one, two, or three, substituents. When two
or more substituents are present in a heterocyclyl group, each
substituent is independently selected. Exemplary substituents for
heterocyclyl group include, but are not limited to, alkyl,
haloalkyl, heteroalkyl, halo, nitro, cyano, optionally substituted
phenyl, optionally substituted heteroaryl, optionally substituted
phenyalkyl, optionally substituted heteroaralkyl, acyl, and the
like. In particular, the term heterocycloalkyl includes, but is not
limited to, tetrahydropyranyl, piperidino, piperazino, morpholino,
pyrrolidino, thiomorpholino, thiomorpholino-1-oxide,
thiomorpholino-1,1-dioxide, and the derivatives thereof. The term
"cycloalkyl" refers to a saturated monovalent cyclic hydrocarbon
moiety of three to seven ring carbons. The cycloalkyl may be
optionally substituted independently with one, two, or three
substituents selected from alkyl, haloalkyl, halo, nitro, cyano,
heteroalkyl, optionally substituted phenyl, optionally substituted
heteroaralkyl, or --C(O)R (where R is hydrogen, alkyl, haloalkyl,
amino, monsubstituted amino, disubstituted amino, hydroxy, alkoxy,
or optionally substituted phenyl). More specifically, the term
cycloalkyl includes, for example, cyclopropyl, cyclopentyl,
cyclohexyl, and the like. The term "heteroaryl" means a monovalent
monocyclic or bicyclic aromatic moiety of 5 to 12 ring atoms
containing one, two, or three ring heteroatoms selected from N, O,
or S, the remaining ring atoms being C. The heteroaryl ring is
optionally substituted independently with one or more substituents,
typically one or two substituents, selected from alkyl, haloalkyl,
heteroalkyl, heterocyclyl, halo, nitro, cyano, carboxy, acyl,
-(alkylene).sub.n-COOR (where n is 0 or 1 and R is hydrogen, alkyl,
optionally substituted phenylalkyl, or optionally substituted
heteroaralkyl), or -(alkylene).sub.n-CONR.sup.aR.sup.b (where n is
0 or 1, and R.sup.a and R.sup.b are, independently of each other,
hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, hydroxyalkyl, aryl,
or R.sup.a and R.sup.b together with the nitrogen atom to which
they are attached form a heterocyclyl ring). More specifically the
term heteroaryl includes, but is not limited to, pyridyl, furanyl,
thiophenyl, thiazolyl, isothiazolyl, triazolyl, imidazolyl,
isoxazolyl, pyrrolyl, pyrazolyl, pyrimidinyl, benzofuranyl,
isobenzofuranyl, benzothiazolyl, benzoisothiazolyl, benzotriazolyl,
indolyl, isoindolyl, benzoxazolyl, quinolyl, isoquinolyl,
benzimidazolyl, benzisoxazolyl, benzothiophenyl, dibenzofuran, and
benzodiazepin-2-one-5-yl, and the like. "Protecting group" refers
to a moiety, except alkyl groups, that when attached to a reactive
group in a molecule masks, reduces or prevents that reactivity.
Examples of protecting groups can be found in T.W. Greene and
P.G.M. Wuts, Protective Groups in Organic Synthesis, 3.sup.rd
edition, John Wiley & Sons, New York, 1999, and Harrison and
Harrison et al., Compendium of Synthetic Organic Methods, Vols. 1-8
(John Wiley and Sons, 1971-1996), which are incorporated herein by
reference in their entirety. Representative hydroxy protecting
groups include acyl groups, benzyl and trityl ethers,
tetrahydropyranyl ethers, trialkylsilyl ethers and allyl ethers.
Representative amino protecting groups include, formyl, acetyl,
trifluoroacetyl, benzyl, benzyloxycarbonyl (CBZ),
tert-butoxycarbonyl (Boc), trimethyl silyl (TMS),
2-trimethylsilyl-ethanesulfonyl (SES), trityl and substituted
trityl groups, allyloxycarbonyl, 9-fluorenylmethyloxycarbonyl
(FMOC), nitro-veratryloxycarbonyl (NVOC), and the like.
"Corresponding protecting group" means an appropriate protecting
group corresponding to the heteroatom (i.e., N, O, P or S) to which
it is attached. "A therapeutically effective amount" means the
amount of a compound that, when administered to a mammal for
treating a disease, is sufficient to effect such treatment for the
disease. The "therapeutically effective amount" will vary depending
on the compound, the disease and its severity and the age, weight,
etc., of the mammal to be treated. "Treating" or "treatment" of a
disease includes: (1) preventing the disease, i.e., causing the
clinical symptoms of the disease not to develop in a mammal that
may be exposed to or predisposed to the disease but does not yet
experience or display symptoms of the disease; (2) inhibiting the
disease, i.e., arresting or reducing the development of the disease
or its clinical symptoms; or (3) relieving the disease, i.e.,
causing regression of the disease or its clinical symptoms. When
describing a chemical reaction, the terms "treating", "contacting"
and "reacting" are used interchangeably herein, and refer to adding
or mixing two or more reagents under appropriate conditions to
produce the indicated and/or the desired product. It should be
appreciated that the reaction which produces the indicated and/or
the desired product may not necessarily result directly from the
combination of two reagents which were initially added, i.e., there
may be one or more intermediates which are produced in the mixture
which ultimately leads to the formation of the indicated and/or the
desired product. The term "polyether" refers to a moiety of the
formula --[O].sub.x--[(CH.sub.2).sub.y--O].sub.z--R.sup.a, where x
is 0 or 1 (x is 0 for R.sup.1); each y is independently an integer
from 2 to 6, typically 2 to 5 and often 2 or 3; z is an integer
from 1 to 20, typically 1 to 10, often 1 to 8, and most often 2 to
6; and R.sup.a is alkyl.
In some embodiments, R.sup.1 is selected from the group consisting
of alkyl, alkenyl, polyether, alkoxy and cycloalkyl; X is selected
from the group consisting of methylene (i.e., CH.sub.2),
C(.dbd.O)--[NR.sup.4].sub.a, C.dbd.S, S.dbd.O, SO.sub.2; R.sup.2 is
selected from the group consisting of heterocycloalkyl and
heteroaryl; R.sup.3 is selected from the group consisting of alkyl,
alkenyl, polyether, alkoxy, cycloalkyl, --NR.sup.5R.sup.6,
--C(.dbd.O)NR.sup.5R.sup.6, and --SO.sub.2NR.sup.5R.sup.6; R.sup.4
is hydrogen, alkyl, or a nitrogen protecting group; each of R.sup.5
and R.sup.6 are independently selected from the group consisting of
hydrogen and alkyl; a is 0 or 1; n=1 to 3; and m=0 to 1.
Yet in other embodiments, R.sup.1 is selected from the group
consisting of C.sub.1-C.sub.10 alkyl. Within these embodiments, in
some instances, R.sup.1 is propyl, butyl, or pentyl.
Still in other embodiments, X is C(.dbd.O)--[NR.sup.4].sub.a.
Within these embodiments, in some instances, a is 1. In other
instances, n is 1. Still in other instances, R.sup.2 is
heterocycloalkyl. Within these instances, in some cases R.sup.2 is
piperidinyl or pyrrolidinyl. Yet in other instances, m is 1. Still
in other instances, R.sup.3 is selected from the group consisting
of alkyl and --C(.dbd.O)NR.sup.5R.sup.6. Within these instances, in
some cases, R.sup.3 is iso-pentyl or --C(.dbd.O)NR.sup.5R.sup.6.
Within such instances, in some cases, R.sup.5 is hydrogen. Still in
other cases, R.sup.6 is alkyl. One particular R.sup.6 is
tert-butyl.
Still in other embodiments, Compound of Formula I is selected from
the group consisting of:
##STR00003##
It should be appreciated that combinations of the various groups
described herein form other embodiments. For example, in one
particularly embodiment R.sup.1 is propyl, butyl, or pentyl; X is
C(.dbd.O)--[NR.sup.4].sub.a, a is 1; n is 1; R.sup.2 is piperidinyl
or pyrrolidinyl; m is 1; R.sup.3 is iso-pentyl or
--C(.dbd.O)NR.sup.5R.sup.6; R.sup.5 is hydrogen; and R.sup.6 is
tert-butyl. In this manner, a wide variety of specific compounds
are encompassed within the scope of the invention.
Another aspect of the invention provides a method for treating a
clinical condition associated with T-type calcium channel
activation, said method comprising administering to a subject in
need of such a treatment a therapeutically effective amount of a
Compound of Formula I.
In some embodiments, said clinical condition associated with T-type
calcium channel activation comprises acute inflammatory pain,
tactile allodynia, diabetic neuropathy, pulmonary hypertension,
chemotherapeutic induced neuropathy, chronic pain, diabetes,
epilepsy, visceral pain, cancer pain, cardiac hypertrophy, or a
combination thereof.
Yet in other embodiments, Compound of Formula I has cannabinoid
receptor, and T-type calcium channel inhibitory activities.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows results in percentage of whole cell current inhibition
of human Cav3.2 (T-type) in response to 10 .mu.M application of the
compound series. For Cav3.2 channels, the holding and test
potentials were respectively -110 and -20 mV.
FIG. 2. Panel A shows representative traces of hCav 3.2 before and
after application of 3 .mu.M compounds 10 and 9 respectively. Panel
B shows dose response relations for compound 9 and 10 block of
hCav3.2 channels. The IC50 from the fit with the Hill equation was
1.48 and 3.68 .mu.M respectively (n=6). Panel C shows effect of 3
.mu.M compound 9 and 10 on the steady state inactivation curve for
Cav3.2 channels. Paned D shows the effect of 3 .mu.M compound 9 and
10 on the current voltage relation for Cav3.2 channels.
FIG. 3. Panels A and B shows effect of increasing doses of
intrathecal compound 9 on the first and second phases of
formalin-induced pain. Panels C and D show the effect of increasing
doses of intraperitoneal compound 9 (on the first and second phases
of formalin-induced pain.
FIG. 4. Panel A shows effect of 30 mg/kg intraperitoneal compound 9
on locomotor activity of wild type mice in the open field test.
Panels B and C shows comparison of effect of 10 .mu.g/i.t.
intrathecal compound 9 on the first and second phases of
formalin-induced pain in wild type and Cav3.2 knockout mice
respectively.
FIG. 5 shows the results of blind analyses of the time course of
treatment of neuropathic mice with vehicle or compound 9.
FIG. 6 shows one particular reaction scheme for producing some of
the compounds of the invention.
DETAILED DESCRIPTION OF THE INVENTION
Many of the known organic molecules that have been shown to
modulate T-type calcium channels have structures similar to
endogenous anandamide-related molecules named lipoamino acids[6,
9]. Lipoamino acids are known to interact with T-type calcium
channels and several are also closely related to
endocannabinoids[6, 9]. Many of these T-type blockers also have
shown to interact with cannabinoid (CB) receptors[6, 9].
The present inventors have previously shown that this mixed
T-type/cannabinoid block has beneficial effects in inducing
analgesia in animal models of inflammatory pain [6, 9]. However,
interactions with CB receptors, particularly CB.sub.1 receptors,
can have side effects that may affect mood and memory, in addition
to their known psychoactive effects. Synthetic T-type calcium
channel antagonists based on substituted piperidines have been
previously disclosed. In addition, the present inventors have
previously synthesized and characterized a series of cannabinoid
compounds with the primary structure bearing a carbazole scaffold
[6, 9]. These compounds produced mixed cannabinoid receptor/T-type
channel blockers that were found to be efficacious in animal models
of inflammatory and neuropathic pain. Interestingly, from
structure-activity relationships (SAR), it was determined that
tertiary amines were important for Cav3.2 block [6, 9] and that the
length of the linker attaching the tertiary amine to the carbazole
scaffold affected binding to CB.sub.1 and CB.sub.2 receptors.[10]
Some of these compounds appeared to preferentially and potently
inhibit T-type channels in-vitro. Accordingly, some aspects of the
invention provide compounds that are effective at blocking T-type
calcium channels.
A wide variety of compounds were synthesized and were screened
using whole-cell voltage clamp techniques to identify the most
potent T-type calcium channel inhibitors. Compounds 9 and 10 were
then characterized using radioligand binding assays to determine
their affinity for CB1 and CB2 receptors. The structure-activity
relationship and optimization studies have led to the discovery of
selective T-type calcium channel blockers, e.g., compound 9. As
used herein, the term "T-type calcium channel blocker" refers to a
compound that can modulate, in particular inhibit or reduce
activation of T-type calcium channels. In some embodiments, the
IC.sub.50 of compounds of the invention for inhibiting T-type
calcium channel is about 10 .mu.M or less, typically about 2 .mu.M
or less, and often about 1 .mu.M or less. The term "about" refers
to .+-.20%, typically .+-.10%, and often .+-.5% of the numeric
value.
In one particular embodiment, Compound 9 was efficacious in
mediating analgesia in mouse models of acute inflammatory pain and
in reducing tactile allodynia in the partial nerve ligation model.
This compound was shown to be substantially ineffective (i.e., no
noticeable activity was observed) in Cav3.2 T-type calcium channel
null mice at therapeutically relevant concentrations and it caused
no significant motor deficits in open field tests. Taken together,
these data reveal a novel class of compounds whose physiological
and therapeutic actions are mediated through block of T-type
calcium channels.
Still other aspects of the invention provide compounds of Formula 1
(FIG. 1) that comprise a carbazole scaffold with an added
heterocyclic bearing a tertiary amine. As can be seen, some of the
compounds of the invention include modified chain length attached
to the nitrogen of the carbazole, the length of the linker between
the amide bond and the heterocycle ring and/or introduction of a
lipophilic moiety attached to the heterocycle. In one embodiment,
the compounds were characterized in-vitro for their ability to
blocking transiently expressed human Cav3.2 (hCav3.2) T-type
calcium channels and tested their affinities for cannabinoid
receptors. Compound 9 was also tested in mouse models of
inflammatory and neuropathic pain, revealing potent analgesia by
virtue of its T-type channel blocking ability.
Synthesis of Compounds of the Invention:
One particular synthesis of the carbazole derivatives is outline in
FIG. 6. Amidation under standard peptide coupling conditions[10] of
N-alkylated carbazole-3-carboxylic derivatives 2 with Boc-protected
amines afforded the desired amide derivatives 3 and 6. Deprotection
of the Boc protecting group in the presence of TFA in
dichloromethane followed by alkylation of the resultant compounds 4
and 8 with N-tert-butyl-2-chloroacetamide provided the
corresponding desired compounds 9, 10, 13, 16, and 19 (Table 1 and
Table 2). Compound 20 was prepared by reductive amination of
compound 4 using 3,3-dimethylbutyraldehyde (Table 2).
##STR00004##
TABLE-US-00001 TABLE 1 Radioligand competitive binding assays (mean
.+-. SEM) for carbazole-based analogues. Values are means of three
experiments run in triplicate with standard deviation. rCB1 hCB2
No. R1 R2 K.sub.i.sup.b (nM) K.sub.i.sup.b (nM) 5 ##STR00005##
##STR00006## n.b. (no observed binding) n.b. 6 ##STR00007##
##STR00008## >5,000 2,957 .+-. 1,362 7 ##STR00009## ##STR00010##
n.b. n.b. 8 ##STR00011## ##STR00012## 283 .+-. 65 2,833 .+-. 1305 9
##STR00013## ##STR00014## >5,000 >5,000 10 ##STR00015##
##STR00016## 15.0 .+-. 6.9 1,968 .+-. 906
##STR00017##
TABLE-US-00002 TABLE 2 Analogues of compound 9: systematic
variation in N-alkyl chain length and in the region occupied by the
heterocycle. No. R1 R2 13 ##STR00018## ##STR00019## 16 ##STR00020##
##STR00021## 19 ##STR00022## ##STR00023## 20 ##STR00024##
##STR00025##
In-Vitro Characterization of the Compound Series:
The compounds was screened using whole-cell voltage clamp
techniques for their ability to mediate tonic block of transiently
expressed hCav3.2 channels (FIG. 1). Next, radioligand binding
assays were used to assess the affinities of these compounds for
both CB.sub.1 and CB.sub.2 receptors (Table 1).
In the course of this work on the structure-activity relationship
for this novel series of T-type channel blockers, the optimal
linker length attached to the carbazole's carbonyl (FIG. 1) was
studied. Various degrees of inhibition of these channels were
observed, with compounds 9 and 10 being some of the most potent
blockers of expressed hCav3.2. These two compounds mediated near
complete inhibition at our standard test concentration of 10 .mu.M
(FIG. 1). Interestingly, both of these compounds are very similar
in structure, with both having a cyclic tertiary amine attached to
the carbazole scaffold (Table 1). It has been shown that this
modification helps confer T-type channel blocking activity onto
various organic molecules[6, 9], in agreement with data presented
here.
As shown in Table 1, compound 10 showed high affinity binding to
CB.sub.1 receptors (15 nM), whereas its affinity for the CB.sub.2
receptor was approximately 100-fold lower (2 Compound 9 however,
did not bind to the two receptors with an affinity less than 5
.mu.M for CB.sub.1 and CB.sub.2 respectively. One of the
differences between compounds 9 and 10 is the elongated chain
attached to the nitrogen of the carbazole in 9 (Table 1). This type
of modification has been shown to alter cannabinoid receptor
binding[6, 9, 10], but it has not been demonstrated whether this
modification affects the interactions of these compounds with
Cav3.2. Among the first series of compounds 5 to 10, replacement of
a piperazine moiety by a methylpiperidine moiety appeared to be the
most optimal for decreasing cannabinoid receptor affinity without
impacting T-type calcium channel block. This difference in affinity
for cannabinoid receptors between compounds bearing a piperazine
moiety compared to a methylpiperidine moiety underscores the
importance of chain length when developing compounds that
selectively target T-type calcium channels over cannabinoid
receptors.
The affect of chain length attached to the carbazole's endocyclic
nitrogen (Table 2, compounds 9, 13 and 16) was also determined.
Among the linear N-1 alkyl chains, a pentyl chain appeared to be
optimal for occupying the T-type cavity, because systematically
decreasing the length from n-pentyl in 9 negatively impacted the
respective Cav3.2 blocking activities. Replacement of the
piperidine ring by a pyrrolidine moiety (19) had a slight negative
effect on Cav3.2 block, probably due to the lack of optimal
ligand-receptor van der Waals contacts. Replacing the amide chain
bored by the piperidine ring by an alkyl chain (20) significantly
decreased the Cav3.2 block.
As can be seen in FIG. 2 panel A, the slight structural
modification in 9 compared to 10 does indeed impact hCav3.2 channel
inhibition. The affinity of 9 versus 10 increased more than
two-fold with the IC.sub.50 of 9 and 10 being 1.48 .mu.M and 3.68
.mu.M respectively (FIG. 2 panel B and Table 1). In addition,
compound 10 shifted the half activation potential of hCav3.2 by -12
mV (FIG. 2 panel C and Table 1). There was no significant effect on
half-inactivation potential (FIG. 2 panel D and Table 1).
Effects of Compound 9 In-Vivo on Acute Pain:
Given the T-type channel blocking property of compound 9, it is
believed that this compound can affect pain transmission in animal
models. Compound 9 was delivered by either intrathecal (i.t.) or
intraperitoneal (i.p.) routes and its effects on both the acute
nociceptive and the slower inflammatory pain phases of the formalin
test were evaluated. One-way ANOVA revealed that i.t. treatment of
mice with compound 9 (1-10 .mu.g/i.t., 20 minutes before)
significantly decreased pain response time in both first (FIG. 3A)
and second (FIG. 3 panel B) phases (61.+-.8% and 76.+-.10%
inhibition, respectively). I.p. treatment of mice with compound 9
(10-100 mg/kg, i.p., 30 minutes prior) also resulted in
significantly (one-way ANOVA) reduced pain response time in both
the first (FIG. 3C) and second (FIG. 3 panel D) phases (47.+-.2%
and 66.+-.48% inhibition, respectively). Importantly, systemic (via
i.p.) treatment with compound 9 (30 mg/kg, i.p.) did not
significantly affect locomotor activity of mice assessed via an
open-field test (FIG. 4 panel A), suggesting that the reduced
response times observed in the previous formalin tests were not due
to altered motor behavior. In order to investigate if the effects
observed for compound 9 were specifically mediated via T-type
channels, a formalin test was performed in Ca.sub.v3.2 null mice
that were treated either with vehicle or with compound 9 (10
.mu.g/i.t.). The Ca.sub.v3.2 null mice exhibited a lower mean
response time when compared to wild-type mice, which is in
agreement with previous data[4, 6]. As indicated in FIG. 4 panels B
and C, they appear to be substantially insensitive to i.t.
treatment with compound 9 (10 .mu.g i.t.) as revealed by two-way
ANOVA, indicating that compound 9 mediates its analgesic effects
specifically via T-type channels.
Effect of Compound 9 on Chronic Neuropathic Pain:
To verify whether compound 9 modulates pain transmission under
neuropathic conditions, mechanical withdrawal thresholds was
analysed in mice with a partial sciatic nerve injury (PNI) and
treated with compound 9 (30 mg/kg, i.p.) 14 days after nerve
injury. As shown in FIG. 5, sciatic nerve injury triggers
mechanical hyperalgesia as confirmed by significant decrease of
mechanical withdrawal thresholds when compared to baselines levels
(Pre-PNI, P<0.001). Two-way ANOVA revealed that systemic (i.p.)
treatment of mice with compound 9 (30 mg/kg, i.p.) significantly
attenuated the mechanical hyperalgesia induced by sciatic nerve
injury when compared with the PNI+Control group for longer than 3
hours after treatment. These data indicate that compound 9
treatment modulates pain transmission and mediates analgesia in
this animal model of chronic neuropathic pain.
TABLE-US-00003 TABLE 3 Biophysical parameters of hCav3.2 calcium
channel in the absence and the presence of compound 10 and 9.
V.sub.0.5 act mV) 3 Vh (mV) 3 IC50 Tonic .mu.M .mu.M (.mu.M) Wt
hCav3.2 -30.0 -53.1 .+-. 1.67 Compound 9 -29.7 -58.2 .+-. 1.43 1.48
.+-. 0.2 Compound 10 -42.0* -58.3 .+-. 1.31 3.68 .+-. 0.5
Discussion:
T-type calcium channels are important contributors to a range of
physiological functions[11] and it is well established that they
play important roles in the afferent pain pathway[2, 4, 5]. Finding
specific and selective blockers of these channels has proven
difficult as many of the well-known T-type channel blockers such as
mibefradil or ethosuximide block other channels, which can then
result in side effects[2]. The present invention provides compounds
with structures similar to some of the endogenous ligands that are
known to interact with T-type channels[6, 9]. In particular,
compounds of the invention include structures that were modified to
reduce their affinity for cannabinoid receptors while attempting to
increase affinity for Cav3.2 channels. Thus, in some embodiments,
compounds of the invention have reduced affinity for cannabinoid
receptors while having increased affinity for Cav3.2 channels
compared to, for example, mibefradil or ethosuximide.
In some embodiments, compounds of the invention are based on
endogenous cannabinoid ligands that targeted both CB receptors and
T-type calcium channels[6, 9]. Using data obtained from these
experiments, additional compounds were designed with an extra
substituted tertiary amine attached to the carbazole scaffold. The
chain length attached to the carbazole to one of the compounds
(compound 9) were then extended to improve its selectivity for
T-type channels over CB receptors[6, 9], e.g., compared to
mibefradil or ethosuximide. These data show that the length of the
linker between the carbazole scaffold and the heterocyclic moiety
is one of the key drug structural determinants that can be
exploited to produce better and more selective T-type channel
inhibitors. The observation that some compounds of the invention's
analgesic actions were abolished upon removal of Cav3.2 channels
indicates that the biological target for compound 9 in the context
of pain signaling is Cav3.2. The potent effects of compound 9 on
pain response in injured wild type animals fits with the notion
that T-type channels play an important role in the afferent pain
pathway[7, 8] and also with a number of previous studies showing
that Cav3.2 channel blockers are efficacious in various pain
models[2, 5].
Data presented herein suggest that using a carbazole scaffold is an
effective strategy for developing potent, selective T-type calcium
channel blockers for therapeutic intervention into inflammatory and
neuropathic pain hypersensitivity. In addition, T-type channels are
also associated with many other disorders, including epilepsy and
cardiac hypertrophy[8], therefore the novel pharmacophores that are
disclosed herein can be used towards treatment of these
disorders.
Additional objects, advantages, and novel features of this
invention will become apparent to those skilled in the art upon
examination of the following examples thereof, which are not
intended to be limiting. In the Examples, procedures that are
constructively reduced to practice are described in the present
tense, and procedures that have been carried out in the laboratory
are set forth in the past tense.
EXAMPLES
In Vitro Receptor Radioligand CB.sub.1 and CB.sub.2 Binding
Studies:
CB.sub.1 and CB.sub.2 radioligand binding data were obtained using
National Institute of Mental Health (NIMH) Psychoactive Drug
Screening Program (PDSP) resources as described earlier[9].
cDNA Constructs:
Human Cav3.2 cDNA construct was obtained from the University of
British Columbia (Vancouver, Canada).
tsA-201 Cell Culture and Transfection:
Human embryonic kidney tsA-201 cells were cultured and transfected
using the calcium phosphate method as described previously.
Transfected cells were then incubated 48 hours at 37.degree. C. and
5% CO.sub.2 and then re-suspended with 0.25% (w/v) trypsin-EDTA
(Invitrogen) and plated on glass coverslips a minimum of 3 to 4
hours before patching.
Electrophysiology:
Whole-cell voltage-clamp recordings from tsA-201 cells were
performed at room temperature 2 to 3 days after transfection.
External recording solution contained (in mM): 114 CsCl, 20 BaC12,
1 MgCl2, 10 HEPES, 10 Glucose, adjusted to pH 7.4 with CsOH.
Internal patch pipette solution contained (in mM): 126.5
CsMeSO.sub.4, 2 MgCl.sub.2, 11 EGTA, 10 HEPES adjusted to pH 7.3
with CsOH. Internal solution was supplemented with 0.6 mM GTP and 2
mM ATP and mixed thoroughly just prior to use. Liquid junction
potentials for the recording solutions were left uncorrected.
Tested compounds were prepared daily from DMSO stocks diluted in
external solution. Using a custom built gravity driven
micro-perfusion system, diluted compounds were then applied rapidly
and locally to the cells. Control vehicle experiments were
performed to ensure that 0.1% DMSO had no effect on current
amplitudes or on the half-activation and half-inactivation
potentials (data not shown). Currents were measured using the
whole-cell patch clamp technique and an Axopatch 200B amplifier in
combination with Clampex 9.2 software (Molecular Devices,
Sunnyvale, Calif.). After establishing whole cell configuration,
cellular capacitance was minimized using the amplifier's built-in
analog compensation. Series resistance was compensated by at least
85% in all experiments. All data were digitized at 10 kHz with a
Digidata 1320 interface (Molecular Devices) and filtered at 1 kHz
(8-pole Bessel filter). Raw and online leak-subtracted data were
both collected simultaneously. In current-voltage relation studies,
the membrane potential was held at -110 mV and cells were
depolarized from -80 to 20 mV in 10 mV increments. For steady-state
inactivation studies, a 3.6 second conditioning pre-pulse of
various magnitude (initial holding at -110 mV), was followed by a
depolarizing pulse to -20 mV. Individual sweeps were separated by
12 seconds to permit recovery from inactivation between
conditioning pulses. The duration of the test pulse was typically
200 ms and the current amplitude obtained from each test pulse was
normalized to that observed at the holding potential of -110
mV.
Animals:
During experiments, all efforts were made to minimize animal
suffering according to the policies and recommendations of the
International Association for the Study of Pain and all protocols
used were approved by the Institutional Animal Care and Use
Committee. For all experiments, either adult male C57BL/6J
(wild-type) or CACNA1H knockout (Cav3.2 null) mice (20-25 g)
purchased from Jackson Laboratories were used. There were a maximum
of five mice per cage (30.times.20.times.15 cm) and access to food
and water was unlimited. Temperature was kept at 23.+-.1.degree. C.
on a 12 h light/dark cycle (lights on at 7:00 a.m.).
Intraperitoneal (i.p.) injections of drugs were a constant volume
of 10 ml/kg body weight. Intrathecal (i.t.) injections used volumes
of 10 .mu.l and were performed using the method described
previously and carried out routinely in our laboratory. All drugs
were dissolved in 1% or less DMSO, whereas control animals received
PBS+1% DMSO. For each test, a different group of mice were used and
only one experiment per mouse was performed. In experiments
examining the action of compound 9 on PNI-induced neuropathy, the
observer was blind to the conditions tested.
Formalin Test:
Before experiments were performed, mice were left to acclimatize
for at least 60 minutes. Animals were then injected intraplantarily
(i.pl.) in the ventral surface of the right hindpaw with 20 .mu.l
of a formalin solution (1.25%) made up in PBS. Following i.pl.
injections of formalin, individual animals were placed immediately
into observation chambers and monitored from 0-5 min (acute
nociceptive phase) and 15-30 min (inflammatory phase). The time
spent licking or biting the injected paw was then considered as a
nociceptive response and recorded with a chronometer. compound 9
was delivered by i.t. (20 minutes prior) or by i.p. (30 minutes
prior) and its effect on both the nociceptive and inflammatory
phases of the formalin test was evaluated.
Open-Field Test:
Animals received compound 9 via i.p. (30 mg/kg) route 30 minutes
before testing and the ambulatory behavior of the treated animals
was assessed in an open-field test as described previously.
Briefly, the apparatus consisted of a wooden box measuring
40.times.60.times.50 cm with a glass front wall. The floor was
divided into 12 equal squares and the entire apparatus was placed
in a sound free room. Animals were placed in the rear left square
and left to explore freely and the number of squares crossed with
all paws (crossing) in a 6 minute timeframe was counted. After each
individual mouse session, the apparatus was then cleaned and dried
with a 10% alcohol solution.
Partial Sciatic Nerve Injury (PNI)-Induced Neuropathic Pain:
Before surgery, mice were anaesthetised with isoflurane (5%
induction, 2.5% maintenance). Partial ligation of the sciatic nerve
was performed by tying the distal 1/3 to 1/2 of the dorsal portion
as previously described. In sham-operated mice, the sciatic nerve
was exposed without ligation and all wounds were closed and treated
with iodine solution. After fourteen days post surgery, mice
received either compound 9 (30 mg/kg, i.p.) treatment or vehicle,
while sham-operated animals received only vehicle (10 ml/kg, i.p.).
Mechanical hyperalgesia was then evaluated in a time-dependant
manner.
Evaluation of Mechanical Hyperalgesia:
Mechanical hyperalgesia responses were recorded immediately before
the surgeries (baselines), 14 days after the surgeries (0) and at
various time points (0.5, 1, 2, 3 h) after treatment. Measurements
were made using a Dynamic Plantar Aesthesiometer (DPA, Ugo Basile,
Varese, Italy). Briefly, individual animals were placed in small
enclosed testing arenas (20 cm.times.18.5 cm.times.13 cm,
length.times.width.times.height) on top of a wire mesh floor and
allowed to acclimate for a period of at least 90 minutes. The DPA
device was then positioned so that the filament was directly under
the plantar surface of the ipsilateral hind paw of the animal and
tested three times per session.
Data Analysis and Statistics:
Data were analyzed using Clampfit 9.2 (Molecular Devices). Origin
7.5 software (Northampton, Mass., USA) was used in the preparation
of all figures and curve fittings. Current-voltage relationships
were fitted with the modified Boltzmann equation:
I=[Gmax*(Vm-Erev)]/[1+exp((V0.5act-Vm)/ka)], where Vm is the test
potential, V0.5act is the half-activation potential, Erev is the
reversal potential, Gmax is the maximum slope conductance, and ka
reflects the slope of the activation curve. Steady-state
inactivation curves were fitted using the Boltzmann equation:
I=1/(1+exp((Vm-Vh)/k)), where Vh is the half-inactivation potential
and k is the slope factor. Dose-response curves were fitted with
the equation y=A2+(A1-A2)/(1+([C]/IC50).sup.n) where A1 is initial
current amplitude and A2 is the current amplitude at saturating
drug concentrations, [C] is the drug concentration and n is the
Hill coefficient. Statistical significance was determined by paired
or unpaired Student's t-tests and one-way or repeated measures
ANOVA, followed by Dunnett's test or Tukey's Multiple Comparison
tests. Significant values were set as indicated in the text and
figure legends. All data are given as means+/-standard errors.
Synthesis of Compounds:
All moisture sensitive reactions were performed in an inert, dry
atmosphere of argon in flame dried glassware. Air sensitive liquids
were transferred via syringe or cannula through rubber septa.
Reagent grade solvents were used for extraction and flash
chromatography. THF was distilled from Na/benzophenone under argon;
dichloromethane (CH.sub.2Cl.sub.2) and chloroform (CHCl.sub.3) were
distilled from CaH.sub.2 under argon. All other reagents and
solvents which were purchased from commercial sources were used
directly without further purification. The progress of reactions
was checked by analytical thin-layer chromatography (Sorbent
Technologies, Silica G TLC plates w/UV 254). The plates were
visualized first with UV illumination followed by charring with
ninhydrin (0.3% ninhydrin (w/v), 97:3 EtOH-AcOH). Flash column
chromatography was performed using prepacked Biotage SNAP
cartridges on a Biotage Isolera One instrument. The solvent
compositions reported for all chromatographic separations are on a
volume/volume (v/v) basis. .sup.1HNMR spectra were recorded at 400
or 500 MHz and are reported in parts per million (ppm) on the
.delta. scale relative to tetramethylsilane as an internal
standard. .sup.13CNMR spectra were recorded at 100 or 125 MHz and
are reported in parts per million (ppm) on the .delta. scale
relative to CDCl.sub.3 (.delta. 77.00). Melting points were
determined on a Stuart melting point apparatus from Bibby
Scientific Limited and are uncorrected. High Resolution mass
spectrometry (HRMS) was performed on a Waters/Micromass LCT-TOF
instrument. All compounds were more than 95% pure.
N-((1-(2-(tert-butylamino)-2-oxoethyl)piperidin-4-yl)methyl)-9-pentyl-9H-c-
arbazole-3-carboxamide (9)
Under nitrogen atmosphere, a mixture of
9-Pentyl-N-(piperidin-4-ylmethyl)-9H-carbazole-3-carboxamide 7[6]
(389 mg, 1.03 mmol), N-tert-butyl-2-chloroacetamide (185 mg, 1.24
mmol), potassium carbonate (427 mg, 3.09 mmol), and potassium
iodide (160 mg, 0.96 mmol) in n-butanol (5 mL) was subjected to
microwave irradiation at 110.degree. C. for 3 h. The mixture was
allowed to cool to room temperature, diluted with DCM (50 mL) and
filtered. The organic solvents were evaporated in vacuo. The
residue was purified on a Biotage.RTM. KP-NH cartridge
(amino-modified silica gel) using cyclohexane/EtOAc in different
proportions to afford the title compound as a light yellow glass.
Yield: 476 mg (94%). .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 8.49
(d, J=1.3 Hz, 1H), 8.01 (d, J=7.7 Hz, 1H), 7.84 (dd, J=8.6, 1.6 Hz,
1H), 7.41 (ddd, J=8.3, 7.1, 1.2 Hz, 1H), 7.33 (d, J=8.2 Hz, 1H),
7.29 (d, J=8.6 Hz, 1H), 7.16 (ddd, J=8.0, 7.1, 1.0 Hz, 1H), 7.00
(br s, 1H), 6.58 (t, J=5.8 Hz, 1H), 4.19 (t, J=7.2 Hz, 2H), 3.34
(t, J=6.5 Hz, 2H), 2.81-2.67 (m, 4H), 2.00 (td, J=11.7, 2.5 Hz,
2H), 1.83-1.74 (m, 2H), 1.71 (br d, J=13.9 Hz, 2H), 1.64-1.48 (m,
1H), 1.31-1.22 (m, 15H), 0.78 (t, J=6.9 Hz, 3H). .sup.13C and DEPT
NMR (101 MHz, CDCl.sub.3) .delta. 169.84 (C.dbd.O), 168.52
(C.dbd.O), 142.33 (C), 141.15 (C), 126.44 (CH), 125.28 (C), 124.81
(CH), 122.96 (C), 122.73 (C), 120.68 (CH), 119.85 (CH), 119.68
(CH), 109.23 (CH), 108.50 (CH), 62.73 (CH.sub.2), 53.89 (CH.sub.2),
50.51 (C), 45.57 (CH.sub.2), 43.37 (CH.sub.2), 36.23 (CH), 30.61
(CH.sub.2), 29.48 (CH.sub.2), 28.93 (CH.sub.3), 28.76 (CH.sub.2),
22.57 (CH.sub.2), 14.08 (CH.sub.3). ESI: m/z 491.2 (M+H).sup.+.
HRMS calcd for C.sub.30H.sub.43N.sub.4O.sub.2 (M+H).sup.+ 491.3386,
found 491.3433.
N-tert-butyl-2-[4-(9-pentyl-9H-carbazole-3-carbonyl)piperazin-1-yl]acetami-
de (10)
Under argon atmosphere, to a solution of tert-Butyl
4-(9-pentyl-9H-carbazole-3-carbonyl)piperazine-1-carboxylate 8[6]
(360 mg, 1.03 mmol) and MeCN (10 mL) was added K.sub.2CO.sub.3 (427
mg, 3.09 mmol), KI (160 mg, 0.96 mmol) and
N-tert-butyl-2-chloroacetamide (185 mg, 1.24 mmol). The reaction
mixture was stirred at reflux for 3 h. The reaction mixture was
cooled, diluted with DCM (30 mL), and filtered. The organic
solvents were evaporated in vacuo. The organic layer was washed
three times with brine (50 mL). The organic layer was then
separated, dried over magnesium sulfate, filtered, and concentrated
in vacuo. The crude product was purified by flash chromatography
eluting with heptane/EtOAc (0-100%) to yield the title compound
(437 mg, 92%) as yellow foam. .sup.1H NMR (400 MHz, CDCl.sub.3)
.delta. 8.19 (d, J=1.24 Hz, 1H), 8.09 (d, J=7.66 Hz, 1H), 7.38-7.55
(m, 4H), 7.23-7.28 (m, 1H), 6.96 (s, 1H), 4.30 (t, J=7.24 Hz, 2H),
3.74 (br. s., 4H), 2.96 (s, 2H), 2.58 (br. s., 4 H), 1.86 (br. s.,
2H), 1.24-1.39 (m, 13H), 0.81-1.01 (m, 3H). .sup.13C NMR (101 MHz,
CDCl.sub.3) d ppm 171.69 (C.dbd.O), 168.71 (C.dbd.O), 141.20 (C),
140.94 (C), 126.30 (CH), 125.60 (C), 125.19 (CH), 122.61 (C),
122.50 (C), 120.54 (CH), 120.14 (CH), 119.43 (CH), 109.07 (CH),
108.51 (CH), 77.44 (CH.sub.2), 77.33 (CH.sub.2), 77.13 (CH.sub.2),
76.81 (CH.sub.2), 62.27 (CH.sub.2), 53.48 (CH.sub.2), 50.64 (C),
43.24 (CH.sub.2), 29.38 (CH.sub.2), 28.83 (CH.sub.2), 28.66
(CH.sub.3), 22.48 (CH.sub.2), 13.98 (CH.sub.3). m/z 463.2 (M+H)+.
HRMS calcd for C.sub.30H.sub.43N.sub.4O.sub.2 (M+H)+ 463.3073,
found 463.2819.
tert-butyl
4-((9-propyl-9H-carbazole-3-carboxamido)methyl)-piperidine-1-ca-
rboxylate (11)
9-propyl-9H-carbazole-3-carboxylic acid[6] (100 mg, 0.40 mmol),
HOBt (62.32 mg, 0.48 mmol), DIPEA (0.13 mL, 0.79 mmol), DMAP (58.12
mg, 0.40 mmol), and EDAC (91.22 mg, 0.40 mmol) were added upon
stirring to DCM (9 mL) under nitrogen. The obtained solution was
cooled down on an ice-water bath and tert-Butyl 4-(aminomethyl)
piperidine-1-carboxylate (119.24 mg, 0.40 mmol) was added in one
portion, and the resulting reaction mixture was then allowed to
warm to room temperature and stirred for 16 h. The solvent was
removed in vacuo, and the obtained residue was extracted with EtOAc
(50 mL). The organic layer was washed three times with brine (25
mL). The organic layer was then separated, dried over magnesium
sulfate, filtered, and concentrated in vacuo. The crude product was
purified by flash chromatography eluting with heptane/EtOAc
(0-100%) to produce the title compound 11 (158.3 mg, 89%) as yellow
oil. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 8.57 (d, J=1.36 Hz,
1H), 8.06 (d, J=7.9 Hz, 1H), 7.89 (dd, J=8.7, 1.67 Hz, 1.11H), 7.44
(ddd, J=8.37, 7.15, 1.2 Hz, 1.1H), 7.31 (d, J=8.2 Hz, 1.0H), 7.27
(d, J=8.7 Hz, 1H), 7.19 (ddd, J=8.06, 7.1, 1.0 Hz, 1H), 6.55 (t,
J=5.8 Hz, 1H), 4.26 (t, J=7.2 Hz, 2H), 3.38 (t, J=6.2 Hz, 2H), 3.10
(d, J=12.1 Hz, 2H), 2.61 (td, J=12.2, 2.5 Hz, 2H), 1.99 (s, 1H),
1.97-1.80 (m, 2H), 1.80-1.70 (m, 3H), 1.45 (s, 9H), 1.22 (qd,
J=12.8, 3.7 Hz, 2H), 0.97 (t, J=7.4 Hz, 3H). .sup.13C (101 MHz,
CDCl.sub.3) .delta. 168.53, 154.77, 142.30, 141.16, 126.42, 125.42,
124.77, 122.97, 122.74, 120.74, 119.83, 119.68, 109.22, 108.53,
79.22, 45.22, 45.39, 36.91, 31.38, 28.41, 22.58, 12.10.
9-Propyl-N-(piperidin-4-ylmethyl)-9H-carbazole-3-carboxamide
(12)
Trifluoroacetic acid (5 mL) was added to a solution of 11 (150 mg,
0.33 mmol) in CH.sub.2Cl.sub.2 (5 mL), and the reaction mixture was
stirred at room temperature for 4 h. The mixture was evaporated,
and the residue was basified with 2 N NaOH. The solution was
extracted with CH.sub.2Cl.sub.2 three times. The extracts were
dried with Na.sub.2SO.sub.4 and evaporated. The crude product was
purified by flash chromatography eluting with heptane/EtOAc
(0-100%) to produce the title compound 12 (95.5 mg, 83%) as dark
yellow oil. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 8.56 (d,
J=1.4 Hz, 1H), 8.05 (d, J=7.9 Hz, 1H), 7.90 (dd, J=8.7, 1.7 Hz,
1.1H), 7.5 (ddd, J=8.4, 7.2, 1.2 Hz, 1.1H), 7.31 (d, J=8.2 Hz,
1.0H), 7.28 (d, J=8.7 Hz, 1H), 7.17 (ddd, J=8.0, 7.1, 1.0 Hz, 1H),
6.58 (t, J=5.8 Hz, 1H), 4.25 (t, J=7.1 Hz, 2H), 3.39 (t, J=6.2 Hz,
2H), 3.09 (d, J=12.1 Hz, 2H), 2.60 (td, J=12.2, 2.5 Hz, 2H), 1.99
(s, 1H), 1.98-1.80 (m, 2H), 1.80-1.71 (m, 3H), 1.23 (qd, J=12.8,
3.7 Hz, 2H), 0.96 (t, J=7.5 Hz, 3H). .sup.13C (101 MHz, CDCl.sub.3)
.delta. 168.51, 142.32, 141.15, 126.40, 125.41, 124.79, 122.99,
122.73, 120.73, 119.82, 119.67, 109.21, 108.50, 46.47, 46.20,
45.37, 36.91, 31.37, 22.59, 12.10.
N-((1-(2-(tert-butylamino)-2-oxoethyl)piperidin-4-yl)methyl)-9-propyl-9H-c-
arbazole-3-carboxamide (13)
Under argon atmosphere, to a solution of 12 (90 mg, 0.26 mmol) and
MeCN (2.5 mL) was added K.sub.2CO.sub.3 (107.8 mg, 0.78 mmol), KI
(43.2 mg, 0.26 mmol) and N-tert-butyl-2-chloroacetamide (47.9 mg,
0.32 mmol). The title compound was prepared according to the
analogous procedure described above for compound 9 to produce the
title compound 13 (121.3 mg, 82%) as dark yellow oil. .sup.1H NMR
(400 MHz, CDCl3) .delta. 8.56 (d, J=1.39 Hz, 1H), 8.05 (d, J=8 Hz,
1H), 7.91 (dd, J=8.7, 1.6 Hz, 1H), 7.44 (ddd, J=8.3, 7.1, 1.2 Hz,
1H), 7.31 (d, J=8.2 Hz, 1H), 7.28 (d, J=8.7 Hz, 1H), 7.17 (ddd,
J=8.1, 7.1, 1.0 Hz, 1H), 6.98 (br s, 1H), 6.58 (t, J=5.8 Hz, 1H),
4.26 (t, J=7.1 Hz, 2H), 3.33 (t, J=6.3 Hz, 2H), 2.82-2.67 (m, 4H),
2.03 (td, J=11.8, 2.7 Hz, 2H), 1.86-1.76 (m, 2H), 1.7 (d, J=13.7
Hz, 2H), 1.64-1.48 (m, 1H), 1.34 (s, 9H), 0.94 (t, J=7 Hz, 3H).
.sup.13C and DEPT NMR (101 MHz, CDCl3) .delta. 169.84 (C.dbd.O),
168.5 (C.dbd.O), 142.34 (C), 141.17 (C), 126.41 (CH), 126.0 (C),
124.79 (CH), 122.94 (C), 122.71 (C), 120.68 (CH), 119.84 (CH),
119.69 (CH), 109.23 (CH), 108.51 (CH), 62.74 (CH.sub.2), 53.9
(CH.sub.2), 50.52 (C), 45.58 (CH.sub.2), 45.35 (CH.sub.2), 36.22
(CH), 30.61 (CH.sub.2), 28.90 (CH.sub.3), 22.76 (CH.sub.2), 12.18
(CH.sub.3). ESI: m/z 463.3 (M+H)+. HRMS calcd for
C.sub.28H.sub.40N.sub.4O.sub.2 (M+H)+, 463.3073 found 463.3089.
Tert-butyl 4-((9-butyl-9H-carbazole-3-carboxamido)methyl)
piperidine-1-carboxylate (14)
Using 9-butyl-9H-carbazole-3-carboxylic acid[6] (100 mg, 0.37
mmol), HOBt (60.54 mg, 0.45 mmol), DIPEA (0.13 mL, 0.75 mmol), DMAP
(54.73 mg, 0.45 mmol), tert-Butyl 4-(aminomethyl)
piperidine-1-carboxylate (112.22 mg, 0.45 mmol) and EDAC (85.85 mg,
0.45 mmol), the title compound was prepared according to the
analogous procedure described above for compound 11, to produce the
title compound 14 (147.3 mg, 85%) as a yellow oil. .sup.1H NMR (400
MHz, CDCl.sub.3) .delta. 8.53 (d, J=1.33 Hz, 1H), 8.04 (d, J=7.82
Hz, 1H), 7.82 (dd, J=8.6, 1.7 Hz, 1H), 7.43 (ddd, J=8.4, 7.14, 1.22
Hz, 1H), 7.31 (d, J=8.22 Hz, 1H), 7.27 (d, J=8.61 Hz, 1H), 7.16
(ddd, J=8.02, 7.1, 1.0 Hz, 1H), 6.57 (t, J=5.8 Hz, 1H), 4.24 (t,
J=7.2 Hz, 2H), 3.37 (t, J=6.2 Hz, 2H), 3.07 (d, J=12.1 Hz, 2H),
2.61 (td, J=12.2, 2.5 Hz, 2H), 2.00 (s, 1H), 1.98-1.80 (m, 2H),
1.80-1.71 (m, 3H), 1.45 (s, 9H), 1.43-1.32 (m, 2H), 1.23 (qd,
J=12.8, 3.7 Hz, 2H), 0.90 (t, J=7.4 Hz, 3H). .sup.13C (101 MHz,
CDCl.sub.3) .delta. 168.50, 154.75, 142.33, 141.16, 126.45, 125.43,
124.80, 122.97, 122.72, 120.73, 119.81, 119.68, 109.21, 108.50,
79.26, 45.47, 43.04, 36.90, 31.36, 28.41, 20.78, 14.13.
9-butyl-N-piperidin-4-ylmethyl)-9H-carbazole-3-carboxamide (15)
Trifluoroacetic acid (5 mL) was added to a solution of 14 (140 mg,
0.30 mmol) in CH.sub.2Cl.sub.2 (5 mL), the title compound was
prepared according to the analogous procedure described above for
compound 12 to produce the title compound 15 (95.7 mg, 88%) as dark
yellow oil. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 8.53 (d,
J=1.3 Hz, 1H), 8.02 (d, J=7.8 Hz, 1H), 7.84 (dd, J=8.6, 1.7 Hz,
1H), 7.41 (ddd, J=8.4, 7.2, 1.2 Hz, 1H), 7.30 (d, J=8.2 Hz, 1H),
7.27 (d, J=8.7 Hz, 1H), 7.17 (ddd, J=8.0, 7.1, 1.0 Hz, 1H), 6.58
(t, J=5.8 Hz, 1H), 4.25 (t, J=7.2 Hz, 2H), 3.39 (t, J=6.2 Hz, 2H),
3.09 (d, J=12.2 Hz, 2H), 2.60 (td, J=12.2, 2.5 Hz, 2H), 1.99 (s,
1H), 1.98-1.80 (m, 2H), 1.80-1.71 (m, 3H), 1.43-1.32 (m, 2H), 1.23
(qd, J=12.8, 3.7 Hz, 2H), 0.90 (t, J=7.4 Hz, 3H). .sup.13C (101
MHz, CDCl.sub.3) .delta. 168.51, 142.32, 141.15, 126.45, 125.41,
124.79, 122.99, 122.73, 120.73, 119.82, 119.67, 109.21, 108.50,
46.47, 46.20, 43.03, 36.91, 31.37, 20.79, 14.12.
N-((1-(2-(tert-butylamino)-2-oxoethyl)piperidin-4-yl)methyl)-9-butyl-9H-ca-
rbazole-3-carboxamide (16)
Under argon atmosphere, to a solution of 15 (90 mg, 0.25 mmol) and
MeCN (2.5 mL) was added K.sub.2CO.sub.3 (103.7 mg, 0.75 mmol), KI
(41.5 mg, 0.25 mmol) and N-tert-butyl-2-chloroacetamide (44.9 mg,
0.30 mmol). The title compound was prepared according to the
analogous procedure described above for compound 9 to produce the
title compound 16 (99.9 mg, 84%) as dark yellow oil. .sup.1H NMR
(400 MHz, CDCl.sub.3) .delta. 8.50 (d, J=1.3 Hz, 1H), 8.01 (d,
J=7.8 Hz, 1H), 7.8 (dd, J=8.6, 1.7 Hz, 1H), 7.4 (ddd, J=8.4, 7.13,
1.22 Hz, 1H), 7.30 (d, J=8.2 Hz, 1H), 7.27 (d, J=8.5 Hz, 1H), 7.16
(ddd, J=8.1, 7.0, 1.0 Hz, 1H), 7.01 (br s, 1H), 6.57 (t, J=5.8 Hz,
1H), 4.24 (t, J=7.2 Hz, 2H), 3.35 (t, J=6.5 Hz, 2H), 2.83-2.68 (m,
4H), 2.00 (td, J=11.7, 2.5 Hz, 2H), 1.82-1.72 (m, 2H), 1.73 (d,
J=13.8 Hz, 2H), 1.63-1.49 (m, 1H), 1.45-1.34 (m, 2H), 1.33 (s, 9H),
0.90 (t, J=6.87 Hz, 3H). .sup.13C and DEPT NMR (101 MHz,
CDCl.sub.3) .delta. 169.83 (C.dbd.O), 168.57 (C.dbd.O), 142.34 (C),
141.14 (C), 126.45 (CH), 125.26 (C), 124.82 (CH), 122.94 (C),
122.73 (C), 120.68 (CH), 119.85 (CH), 119.68 (CH), 109.21 (CH),
108.50 (CH), 62.71 (CH.sub.2), 53.90 (CH.sub.2), 50.50 (C), 45.59
(CH.sub.2), 43.00 (CH.sub.2), 36.26 (CH), 30.66 (CH.sub.2), 29.43
(CH.sub.2), 28.92 (CH.sub.3), 20.74 (CH2), 14.10 (CH3). ESI: m/z
477.2 (M+H)+. HRMS calcd for C.sub.29H.sub.41N.sub.4O.sub.2 (M+H)+
477.3229, found 477.3217.
Tert-butyl
4-((9-pentyl-9H-carbazole-3-carboxamido)methyl)pyrrolidine-1-ca-
rboxylate (17)
Using 9-pentyl-9H-carbazole-3-carboxylic acid (100 mg, 0.36 mmol),
[6] HOBt (57.81 mg, 0.43 mmol), DIPEA (0.12 mL, 0.71 mmol), DMAP
(52.29 mg, 0.43 mmol), (R)-3-(Aminomethyl)-1-Boc-pyrrolidine (85.66
mg, 0.43 mmol) and EDAC (82.02 mg, 0.43 mmol), the title compound
was prepared according to the analogous procedure described above
for compound 11 using the amide coupling protocol, to produce the
title compound 17 (141.8 mg, 86%) as a yellow oil. .sup.1H NMR (400
MHz, CDCl.sub.3) .delta. 8.58 (d, J=1.4 Hz, 1H), 8.02 (d, J=7.8 Hz,
1H), 7.83 (dd, J=8.6, 1.7 Hz, 1H), 7.42 (ddd, J=8.4, 7.14, 1.23 Hz,
1H), 7.31 (d, J=8.22 Hz, 1H), 7.28 (d, J=8.61 Hz, 1H), 7.17 (ddd,
J=8.1, 7.10, 1.0 Hz, 1H), 6.57 (t, J=5.84 Hz, 1H), 4.16 (t, J=7.2
Hz, 2H), 3.22-3.12 (m, 2H), 2.34-2.21 (m, 2H), 2.12-1.98 (m, 1H),
1.81-1.67 (m, 3H), 1.45 (s, 9H), 1.38-1.20 (m, 4H), 0.80 (t, J=6.9
Hz, 3H). .sup.13C (101 MHz, CDCl.sub.3) .delta. 168.56, 154.75,
142.32, 141.13, 126.47, 125.25, 124.82, 122.92, 122.72, 120.69,
119.85, 119.68, 109.22, 108.50, 79.27, 54.28, 45.50, 39.78, 38.75,
36.26, 30.68, 29.45, 28.42, 20.75, 14.10.
9-Pentyl-N-(pyrrolidin-4-ylmethyl)-9H-carbazole-3-carboxamide
(18)
Trifluoroacetic acid (5 mL) was added to a solution of compound 17
(135 mg, 0.29 mmol) in CH.sub.2Cl.sub.2 (5 mL), the title compound
was prepared according to the analogous procedure described above
for compound 12 to produce the title compound 18 (90.7 mg, 86%) as
yellow oil. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 8.57 (d,
J=1.4 Hz, 1H), 8.03 (d, J=7.8 Hz, 1H), 7.84 (dd, J=8.6, 1.7 Hz,
1H), 7.41 (ddd, J=8.4, 7.13, 1.22 Hz, 1H), 7.32 (d, J=8.22 Hz, 1H),
7.27 (d, J=8.61 Hz, 1H), 7.17 (ddd, J=8.1, 7.10, 1.0 Hz, 1H), 6.57
(t, J=5.84 Hz, 1H), 4.16 (t, J=7.2 Hz, 2H), 3.22-3.13 (m, 2H),
2.34-2.22 (m, 2H), 2.12-1.99 (m, 1H), 1.82-1.68 (m, 3H), 1.38-1.20
(m, 4H), 0.82 (t, J=6.9 Hz, 3H). .sup.13C (101 MHz, CDCl.sub.3)
.delta. 168.55, 142.33, 141.14, 126.46, 125.24, 124.82, 122.93,
122.72, 120.69, 119.85, 119.68, 109.22, 108.50, 54.27, 45.50,
39.79, 38.76, 36.26, 30.68, 29.44, 20.76, 14.11.
N-((1-(2-(tert-butylamino)-2-oxoethyl)pyrrolidin-4-yl)methyl)-9-pentyl-9H--
carbazole-3-carboxamide (19)
Under argon atmosphere, to a solution of compound 18 (85 mg, 0.23
mmol) and MeCN (2.5 mL) was added K.sub.2CO.sub.3 (95.4 mg, 0.69
mmol), KI (38.1 mg, 0.69 mmol) and N-tert-butyl-2-chloroacetamide
(41.9 mg, 0.28 mmol). The title compound was prepared according to
the analogous procedure described above for compound 10 to produce
the title compound 19 (96.2 mg, 88%) as dark yellow oil. .sup.1H
NMR (400 MHz, CDCl.sub.3) .delta. 8.58 (d, J=1.4 Hz, 1H), 8.02 (d,
J=7.8 Hz, 1H), 7.83 (dd, J=8.6, 1.7 Hz, 1H), 7.41 (ddd, J=8.4,
7.13, 1.22 Hz, 1H), 7.31 (d, J=8.22 Hz, 1H), 7.27 (d, J=8.61 Hz,
1H), 7.16 (ddd, J=8.1, 7.10, 1.0 Hz, 1H), 7.01 (br s, 1H), 6.56 (t,
J=5.84 Hz, 1H), 4.16 (t, J=7.2 Hz, 2H), 3.22-3.11 (m, 4H),
2.34-2.22 (m, 2H), 2.11-1.99 (m, 1H), 1.81-1.67 (m, 3H), 1.39-1.22
(m, 16H), 0.83 (t, J=6.9 Hz, 3H). .sup.13C and DEPT NMR (101 MHz,
CDCl.sub.3) .delta. 169.83 (C.dbd.O), 168.57 (C.dbd.O), 142.34 (C),
141.14 (C), 126.45 (CH), 125.26 (C), 124.82 (CH), 122.94 (C),
122.73 (C), 120.68 (CH), 119.85 (CH), 119.68 (CH), 109.21 (CH),
108.50 (CH), 63.41 (CH.sub.2), 60.41 (CH.sub.2), 55.56 (CH.sub.2),
50.89 (C), 47.36 (CH.sub.2), 44.07 (CH.sub.2), 36.67 (CH), 30.46
(CH.sub.2), 29.53 (CH.sub.2), 28.88 (CH.sub.3), 20.58 (CH.sub.2),
14.14 (CH.sub.3). ESI: m/z 477.5 (M+H)+. HRMS calcd for
C.sub.29H.sub.41N.sub.4O.sub.2 (M+H)+477.3229, found 477.3251.
N-{[1-(3,3-dimethylbutyl)piperidin-4-yl]methyl}-9-pentyl-9H-carbazole-3-ca-
rboxamide (20)
9-Pentyl-9H-carbazole-3-carboxylic acid[6] (100 mg, 0.27 mmol) was
combined with cyanoborohydride (83.3 mg, 1.35 mmol/g),
3,3-dimethylbutyraldehyde (0.06 mL, 0.31 mmol) and suspended in
anhydrous CH.sub.2Cl.sub.2 (2 mL). The mixture was stirred at room
temperature overnight. The reaction mixture was diluted with DCM
(30 mL), and organic layer was washed three times with brine (50
mL). The organic layer was then separated, dried over magnesium
sulfate, filtered, and concentrated in vacuo. The crude product was
purified by flash chromatography eluting with heptane/EtOAc
(0-100%) to yield the title compound (88.4 mg, 71%). 1H NMR (400
MHz, CDCl.sub.3) .delta. 8.51 (d, J=1.3 Hz, 1H), 8.04 (d, J=7.7 Hz,
1H), 7.86 (dd, J=8.6, 1.6 Hz, 1H), 7.42 (ddd, J=8.3, 7.1, 1.2 Hz,
1H), 7.35 (d, J=8.2 Hz, 1H), 7.29 (d, J=8.6 Hz, 1H), 7.15 (ddd,
J=8.0, 7.1, 1.0 Hz, 1H), 7.01 (br s, 1H), 6.59 (t, J=5.8 Hz, 1H),
4.20 (t, J=7.2 Hz, 2H), 3.36 (t, J=6.5 Hz, 2H), 2.69-2.54 (m, 4H),
2.01 (td, J=11.7, 2.5 Hz, 2H), 1.83-1.74 (m, 4H), 1.64-1.48 (m,
1H), 1.33-1.23 (m, 8H), 0.91 (s, 9H), 0.77 (t, J=6.9 Hz, 3H). 13C
and DEPT NMR (101 MHz, CDCl3) .delta. 168.76 (C.dbd.O), 142.47 (C),
141.23 (C), 126.67 (CH), 125.48 (C), 124.18 (CH), 122.76 (C),
122.53 (C), 120.68 (CH), 119.85 (CH), 119.68 (CH), 109.23 (CH),
108.50 (CH), 53.89 (CH.sub.2), 52.31 (CH.sub.2), 45.67 (CH.sub.2),
43.42 (CH.sub.2), 40.22 (CH.sub.2), 36.21 (CH), 30.54 (CH.sub.2),
30.12 (C), 29.42 (CH.sub.2), 29.34 (CH.sub.3), 28.71 (CH.sub.2),
22.56 (CH.sub.2), 14.03 (CH.sub.3). ESI: m/z 462.3 (M+H)+. HRMS
calcd for C.sub.30H.sub.43N.sub.3O (M+H)+462.3484, found
462.3452.
The foregoing discussion of the invention has been presented for
purposes of illustration and description. The foregoing is not
intended to limit the invention to the form or forms disclosed
herein. Although the description of the invention has included
description of one or more embodiments and certain variations and
modifications, other variations and modifications are within the
scope of the invention, e.g., as may be within the skill and
knowledge of those in the art, after understanding the present
disclosure. It is intended to obtain rights which include
alternative embodiments to the extent permitted, including
alternate, interchangeable and/or equivalent structures, functions,
ranges or steps to those claimed, whether or not such alternate,
interchangeable and/or equivalent structures, functions, ranges or
steps are disclosed herein, and without intending to publicly
dedicate any patentable subject matter. All references cited herein
are incorporated by reference in their entirety.
CITED REFERENCES
(1) Jagodic, M. M., et al., Cell-specific alterations of T-type
calcium current in painful diabetic neuropathy enhance excitability
of sensory neurons. J Neurosci, 2007. 27(12): p. 3305-16. (2)
Barton, M. E., E. L. Eberle, and H. E. Shannon, The
antihyperalgesic effects of the T-type calcium channel blockers
ethosuximide, trimethadione, and mibefradil. Eur J Pharmacol, 2005.
521(1-3): p. 79-85. (3) Bourinet, E., et al., Silencing of the
Cav3.2 T-type calcium channel gene in sensory neurons demonstrates
its major role in nociception. EMBO J, 2005. 24(2): p. 315-24. (4)
Choi, S., et al., Attenuated pain responses in mice lacking
Ca(V)3.2 T-type channels. Genes Brain Behav, 2007. 6(5): p. 425-31.
(5) Dogrul, A., et al., Reversal of experimental neuropathic pain
by T-type calcium channel blockers. Pain, 2003. 105(1-2): p.
159-68. (6) Gadotti, V. M., et al., Analgesic effect of a mixed
T-type channel inhibitor/CB2 receptor agonist. Mol Pain, 2013. 9:
p. 32. (7) Jagodic, M. M., et al., Upregulation of the T-type
calcium current in small rat sensory neurons after chronic
constrictive injury of the sciatic nerve. J Neurophysiol, 2008.
99(6): p. 3151-6. (8) Obradovic, A., et al., CaV3.2 T-Type Calcium
Channels in Peripheral Sensory Neurons Are Important for
Mibefradil-Induced Reversal of Hyperalgesia and Allodynia in Rats
with Painful Diabetic Neuropathy. PLoS One, 2014. 9(4): p. e91467.
(9) You, H., et al., Functional characterization and analgesic
effects of mixed cannabinoid receptor/T-type channel ligands. Mol
Pain, 2011. 7: p. 89. (10) Petrov, R. R., et al., Mastering
tricyclic ring systems for desirable functional cannabinoid
activity. Eur J Med Chem, 2013. 69: p. 881-907. (11) Marger, F., et
al., T-type calcium channels contribute to colonic hypersensitivity
in a rat model of irritable bowel syndrome. Proc Natl Acad Sci USA,
2011. 108(27): p. 11268-73.
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